The field concerns purification of serum albumins, particularly human serum albumins.
Human serum albumin (HSA) is the major protein component of plasma. The primary function of albumin in plasma is maintenance of the colloid osmotic pressure within the blood vessel. Furthermore, the protein acts as a carrier of several ligands, for instance bilirubin and fatty acids. (See reviews by F. Rothstein, V. M. Rosenoer and W. L. Hughes in Albumin Struct. Funct. Uses (1977) 7-25; U. Kragh-Hansen, Pharmacol. Rev. (1981) 33:17-53; T. Peters Jr., Adv. Prot. Chem. (1985) 37:161-245).
Purified serum albumin is indicated for the prevention and treatment of hypovolemic shock, in conditions where there is severe hypoalbuminemia, as an adjunct in haemodialysis and in cardiopulmonary bypass procedures and in conjunction with exchange transfusion in the treatment of neonatal hyperbilirubinemia.
Since large amounts of serum albumin are necessary for therapy and the source of serum albumin (plasma) is limited, other techniques have been sought to produce HSA in large quantities. Successes have been reported in the production of HSA by fermentation using transformed microorganisms or cell lines made by recombinant DNA techniques. See, for example, EP-A-0073646.
However, one of the major problems in the purification of serum albumin produced by fermentation using transformed cells is the presence of contaminating components from the growth medium (fermentation broth) or cell lysate, which have to be removed in order to obtain purified, homogeneous serum albumin.
In EP-A-0361991 the purification of HSA produced with transformed yeast, using techniques known in the art, yields a product of more than 99% purity. For a pharmaceutical preparation a higher purity is desirable.
Recently a process for the purification of serum albumins based on a process in three steps was disclosed in EP-A-0319067. This process starting with an alkaline precipitation step followed by anion-exchange chromatography and finally affinity chromatography yields a product with a good yield and purity. In EP-A-0319067 a BrCN-activated Sepharose 4B support was mentioned which was prepared according to the method described by Wichman and Andersson (1974). For industrial use the affinity matrix based on BrCN-activated sepharose has two major draw-backs.
First, the isourea linkage (M. Wilchek, T. Miron and J. Kohn in: Methods in Enzymology (1984) 104:3, W. B. Jakoley Ed., Academic Press, London) resulting from the reaction with the primary alkylamine (spacer) has a positive charge under physiological conditions. This charged spacer shows anion-exchange like characteristics that might interfere with the biospecific adsorption. A second disadvantage is the limited stability of the isourea linkage under slightly alkaline conditions (C. M. Yang and G. T. Tsao in: Ad. Biochem. Engin. (1982) 25:19, A. Fiechter Ed., Springer Verlag, Berlin-Heidelberg) which enables the use of this matrix under sanitizing conditions (0.1-2.0 M NaOH) necessary for the production of pharmaceutical products.
Therefore, there is a great need for a practical process for large-scale purification of human serum albumin with a high recovery and a very high purity.
Human serum albumin, produced by a transformed host, is purified by ion-exchange chromatography, followed by affinity chromatography employing a lipophilic surface immobile phase comprising a carrier coupled to a 2-mercapto or 2-hydroxy C4-C14 alkanoic acid, or salt or ester thereof. The serum albumin degradation products present at the end of the fermentation which strongly resemble the mature, intact albumin are selectively removed by the fatty acid affinity chromatography applied. High recovery and extremely high purity are achieved.